4 replies to this topic

[katenkak]

Topic says its all. I need to transfect mouse cerebellar and/or hippocampal neurons with plasmid or siRNA. What works best with highest survival rate? I have tried Amaxa electroporation-neurons look ugly and don't survive. When I tried AMaxa with plasmid, neurons survived for 3 days and died. I try with Lipofectamine 2000 and see that during 4 hours of incubation with Lipofectamine, many neurons die and processes of remaining neurons get disintegrated. Moreover efficiency of transfection is close to 1% contra promised 25-20% . Please help me if you have a protocol!!!! Thank you!

[HBImolbiol]

katenkak, on 23 March 2011 - 01:43 AM, said:

Topic says its all. I need to transfect mouse cerebellar and/or hippocampal neurons with plasmid or siRNA. What works best with highest survival rate? I have tried Amaxa electroporation-neurons look ugly and don't survive. When I tried AMaxa with plasmid, neurons survived for 3 days and died. I try with Lipofectamine 2000 and see that during 4 hours of incubation with Lipofectamine, many neurons die and processes of remaining neurons get disintegrated. Moreover efficiency of transfection is close to 1% contra promised 25-20% . Please help me if you have a protocol!!!! Thank you!

You could try a viral vector such as Adeno associated virus (serotype 9) or lentivirus.

[katenkak]

1) Do you know a best vector for lentiviral expression in neurons?
2) I try to find a non-viral protocol, since according to local rules, I can't take live cells outsinde og class 2 area, but only fixed. I need live neurons for real-time measurements.

Edited by katenkak, 23 March 2011 - 07:36 AM.

[swimmer]

You may try to find sth. from this paper.
http://www.nature.co...bs/ncb1881.html

Edited by swimmer, 20 June 2011 - 06:52 AM.

[labrat612]

Have you tried Calcium-phosphate method? It's tedious and time-consuming but it does give descent results.


~Labrat612